The present invention relates to vasoactive amine binding molecules (VABMs) and their use in the regulation of the action of vasoactive amines. The invention in particular relates to VABMs which are derived from parasite proteins or derivatives thereof. The present invention also relates to the detection and quantification of vasoactive amines and to the control of diseases and injury caused by parasites in animals and humans, especially those caused by ectoparasites of domestic animals. It further relates to the use of vasoactive binding molecules in the treatment of diseases and allergies. The present invention also relates to the use of recombinant DNA technology to produce VABMs.
Vasoactive amines such as histamine and serotonin are mediators of inflammation and regulators of certain physiological processes in animals, including humans. Histamine is present in the secretory granules of mast cells and basophils and is,formed by decarboxylation of histidine. It is also present in ergot and plants and may be synthesised synthetically from histidine or citric acid.
The main actions of histamine in humans are stimulation of gastric secretion, contraction of most smooth muscle, cardiac stimulation, vasodilation and increased vascular permeability. In addition to its regulatory role in immune reactions and inflammatory processes, histamine also modulates the production of many cytokines in the body (including those that regulate inflammation) and can interfere with the expression of cytokine receptors. Furthermore, histamine promotes wound healing.
The main pathophysiological roles of histamine are as a stimulant of gastric acid secretion and as a mediator of type I hypersensitivity reactions such as urticaria and hay fever. Histamine or its receptors may also be involved either directly or indirectly in autoimmune disease, e.g. arthritis, and in tumour growth (Falus (1994) Histamine and Inflammation, R. G. Landes Co., Austin; pp.139).
Histamine produces its actions by an effect on specific histamine receptors which are of three main types, H1, H2 and H3, distinguished by means of selective antagonist and agonist drugs. Histamine H1 and H2 receptor antagonists have clinical uses but at present histamine H3 receptor antagonists are used mainly as research tools.
H1 receptor antagonists (antihistamines) are widely used for treating allergic reactions including allergic rhinitis (hay-fever), urticaria, insect bites and drug hypersensitivities. Drugs that lack sedative or muscarinic-receptor antagonist activities are preferred. H1 receptor antagonists are also used as anti-emetics for the prevention of motion sickness or other causes of nausea including severe morning sickness. Muscarinic-receptor antagonist actions of some antihistamines probably contribute to efficacy but also cause side effects. Some H1 receptor antagonists are fairly strong sedatives and may be used for this action.
There are numerous undesirable effects of H1 receptor antagonists. When used for purely antihistamine actions, all the CNS effects are unwanted. When used for their sedative or anti-emetic actions, some of the CNS effects such as dizziness, tinnitus and fatigue are unwanted. Excessive doses can cause excitation and may produce convulsions in children. The peripheral antimuscarinic actions are always undesirable. The commonest of these is dryness of the mouth, but blurred vision, constipation and retention of urine can also occur. Unwanted effects not related to the drugs"" pharmacological actions are also seen. Thus gastro-intestinal disturbances are fairly common while allergic dermatitis can follow topical application of these drugs.
H2 receptor antagonists are frequently used as inhibitors of gastric acid secretion. They are used as the drugs of choice in the treatment of peptic ulcer, as second line drugs in the treatment of Zollinger-Ellison syndrome and for treating reflux oesophagitis. Unwanted effects have been reported that include diarrhoea, dizziness, muscle pains, transient rashes and hyper-gastrinaemia. Some H2 receptor antagonists can cause gynaecomastia in men and confusion in the elderly.
Besides these unwanted side effects, some histamine antagonists are troublesome if taken with alcohol or with drugs. For example, the antihistamine Seldane used in combination with antibiotics and antifungals may cause life-threatening side effects.
Drugs used to control the actions of histamine are not always effective. The reasons why they may have limited efficacy may relate to the specificity of these drugs for only a subclass of histamine-receptors, particularly when certain conditions require interference with a larger spectrum of receptors. Histamine binding molecules (HBMs) would compete for histamine binding with all receptors and may thus be more suited for treating certain conditions.
The hormone serotonin (also known as 5-hydroxytryptamine) is both a vasoconstrictor and a neurotransmitter. It can also increase vascular permeability, induce dilation of capillaries and cause the contraction of nonvascular smooth muscle. Serotonin is present in the brain and intestinal tissues and is produced by the pineal gland and by blood platelets. Pathological aspects related to serotonin include abnormal blood pressure, migraine, psychological disorders, respiratory disease and coronary heart disease. Serotonin agonists and antagonists are used to treat some of these disorders, but again often have undesirable side-effects.
There is thus a great need for effective antagonists of vasoactive amines that do not generate the side-effects that detract from their applicability to the treatment of human and animal disorders.
There is also a need for the quantification of histamine in, for example, food products, various body fluids (e.g. plasma or urine) or cell culture supernatants to monitor the effects of certain allergens, for example, or to point to a specific antagonistic therapy for an allergic reaction. Currently used systems (radioimmunoassays and ELISAs) utilize antibodies against histamine or against histamine-derivatives. However, histamine is not very immunogenic, making it hard to raise high-affinity antibodies against it, and most of the quantitation systems used today are not very sensitive or require modification of the histamine to be measured (for example by acylation or methylation).
The use of HBMs to replace antibodies in assays like these would provide a highly sensitive system to measure unmodified histamine. Another advantage of HBMs over anti-histamine antibodies is that they can be used as research tools for the removal of free (unbound) histamine from, for example, cell cultures when studying certain biological processes. Due to the presence of antibody receptors on most cells, antibodies might interfere with the normal functioning of these cells.
It is known that blood-feeding ectoparasites, such as ticks, produce numerous bioactive proteins that immunomodulate the host response to parasite feeding and thereby promote parasite blood-feeding. Such immunomodulatory proteins include immunoglobulin-binding proteins (IGBPs) that are produced in the haemolymph and saliva of ticks and bind to vertebrate host immunoglobulins (Wang and Nuttall (1995) Parasite Immunology 17:517-514). They also include salivary nitric oxide-carrying haeme protein (nitrophorin) of the triatome bug Rhodnius prolixus, which, in addition to carrying nitric oxide, can also bind histamine (Ribeiro and Walker (1994) J. Exp. Med. 180:2251-2257). Immunomodulatory proteins are also produced by other blood-feeding parasites, such as mosquitoes and leeches, and venom-producing animals such as snakes and spiders.
We have found that blood-feeding ectoparasites, for example ticks, produce proteins capable of binding to vasoactive amines, particularly histamine and serotonin. These proteins are hereafter referred to as vasoactive amine binding proteins (VABPs).
We have isolated from ticks four VABPs which are named herein as MS-HBP1, FS-HBP1, FS-HBP2 and D.RET6 and which are closely related to each other. These proteins are entirely novel and show no significant similarity to any previously described protein. The DNA sequences encoding these proteins or fragments thereof can be used to isolate other related proteins in the same family from the same or different species.
The present invention provides a vasoactive amine binding protein (VABP) that specifically binds to vasoactive amines with a dissociation constant of less than 10xe2x88x927M and which belongs to the same protein family as MS-HBP1, PS-HBP1, FS-HBP2 and D.RET6.
A protein is considered to belong to this family if 40% or more of the amino acids that are completely conserved as identical residues in the alignment of the four VABPs alone, are still completely conserved as identical residues if the protein is included in the alignment, the alignments being obtained using GCG""s pileup command (Program manual for the Wisconsin package, 1994; gap creating penalty=2.50; gap extension penalty=0.05). Also included as a member of the VABP family are those proteins from haematophagous arthropods that bind histamine with an affinity characterised by a dissociation constant less than 10xe2x88x927M and contain the sequence motifs D/E A W K/R (SEQ ID NO:23) and Y/C E/D L/I W (SEQ ID NO:24).
The VABPs of the present invention include natural biological variants (e.g. allelic variants or geographical variations within the species from which the VABPs are derived).
The present invention also includes functionally-equivalent derivatives and fragments of the vasoactive amine binding proteins, or of proteins belonging to the same protein family as the VABPs. The VABPs, derivatives and fragments of the present invention are hereafter referred to as vasoactive amine binding molecules (VABMs).